Institut für Lebensmittelchemie

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Biochemical investigations on genetically modified oil crops
(2009) Mekawi, Enas; Schwack, Wolfgang
The main purpose of this study was to develop a method of purification and characterization of Cry1Ab isolated from MON810 genetically modified maize. The second object was to study the effect of the genetic modification of MON810 and high-oleic sunflower on the oil composition. Therefore, the following investigations were performed: (1) Quantification of Cry1Ab toxin in different corn plant parts. (2) Development of a suitable method for purification of Cry1Ab from MON810. (3) Establishment of characterization method for Cry1Ab by mass spectrometry with regard to high peptide sequence coverage. (4) Evaluation of the effect of genetic modification on the oil composition compared with the conventional traits. The following results were obtained: Screening of Cry1Ab by ELISA is the most predominant technique for determination of Cry toxin content in plants. The determination of the toxin concentration resulted in highest levels for leaves (26.8 µg/g dry matter), while it was 1.5, and 1.0 µg/g for stalks and grains respectively. In our study, toxin content in leaves was about six times higher than in a previous study. There are no data available for the dry weight content of Bt toxins in stalk and grain, which could be compared to the obtained results. Although MON810 maize is one of the major genetically modified crops, informations on the character of the Cry1Ab purified from the MON810 maize is still limited, although such data are important for safety assessment studies. To my best knowledge, this study is the first investigation charactizing Cry1Ab toxin isolated from MON810 maize. The results of the present investigation indicated that the separation of the Cry1Ab protein from MON810 leaf extracts by HPLC techniques was not efficient. MALDI-TOF analyses showed that the major component separated with Cry1Ab was β-D glucosidase, which may be due to resembled isoelectric points. However, immuno-affinity purification using self-prepared affinity columns was very efficient to isolate pure Cry1Ab from MON810. The characterization of purified Cry1Ab was successfully done by SDS-PAGE, Western blot analysis and MS techniques. MALDI-TOF MS analyses were useful for component screening of Cry1Ab. Results showed that Cry1Ab is subjected to truncation by plant proteases into a core toxin with approximately 69 kDa. LC(ESI)-MS/MS gave a higher sequencing coverage of Cry1Ab (73 % of peptide sequence) compared to MALDI-TOF analysis (41% of peptide sequence). Further studies revealed that Cry1Ab had no detectable potential carbohydrates which might be covalently linked to the protein. The capillary electrophoresis technique was used for determination of the Cry1Ab purified from MON810 maize and proved to be a suitable method for determination of the Cry1Ab, but it was not successful for the detection of very low quantities (less than 0.03 mg/ml). Peptide mapping is one of the most powerful tools for protein identification and characterization. The use of HPTLC with the relatively new plates (ProteoChrom) was identified as a convenient tool for peptide mapping as compared to capillary electrophoresis, especially if put into consideration that HPTLC is less costly than capillary electrophoresis. The HPTLC method was able to resolve 13 peptides, while capillary electrophoresis resolved 19 peptides, obtained from the digested Cry1Ab toxin. Concerning lipid analyses, fatty acids and sterols were determined by gas chromatography, tocopherols by HPLC. For the determination of phospholipids, an HPTLC method was developed, resulting in lower detection limits than reported in previous studies. The present study proved that the genetic modification did not significantly affect the contents of fatty acids, sterols, tocopherols and phospholipids in transgenic maize oil. Apart from the increased amount of oleic acid in high-oleic sunflower oil, the genetic modification in sunflower did not produce unexpected effects on the oil composition. Therefore, with regard to the oil composition, both oils from genetically modified plants will be as safe as conventional oil types.
Determination of organophosphorus and carbamate insecticides in food samples by high-performance thin-layer chromatography multi-enzyme inhibition assay
(2011) Akkad, Rami; Schwack, Wolfgang
In terms of effect-directed analysis, esterase inhibitor assays allow a rapid and selective detection of insecticidal organophosphates and carbamates in food and environmental samples. With consideration to the toxicological mechanism of action of these insecticides, cholinesterases of different origin were used in different test formats, as microtiterplate assays, in test strip formats, as biosensors or coupled to thin-layer chromatography (bio-autography). Instead of cholinesterases, Ingrid Walz (PhD thesis, University of Hohenheim, 2008) introduced rabbit liver esterase (RLE), Bacillus subtilis (BS2)-esterase and cutinase (CUT) from Fusarium solani pisi for a multi-enzyme microtiterplate assay. In particular, RLE and BS2 proved to be much more sensitive than chloinesterases for the detection of inhibitors, while CUT displayed oneself by special tolerance for matrix components from fruits. This multi-enzyme assay was successfully transferred onto high-performance thin-layer chromatography (HPTLC). With the insecticide examples of carbofuran, malaoxon and paraoxon as weak, medium and strong inhibitors, HPTLC-enzyme inhibition (HPTLC-EI) assay conditions were optimized concerning enzyme concentrations, incubation times and substrate reactions. In the presence of the substrate α-naphthyl acetate/Fast Blue Salt B leads to colourless inhibitor zones are obtained on a purple background, which can be sensitively quantified by scanning at 533 nm. The limits of detection for paraoxon were determined to 1.3, 1.2, and 540 pg/zone for RLE, BS2, and CUT, respectively. Malaoxon was detectable up to 7.9, 7.4 and 760 ng/zone, while the limits of detection for carbofuran were at 33, 54 and 1420 ng/zone.
Einfluss moderner Pflanzenschutzmittel auf die Mobilität von POP-belasteten Agrarflächen am Beispiel von DDT : ein Feldversuch
(2023) Neitsch, Julia Simone; Vetter, Walter
Due to their recalcitrance, the chloropesticide DDT and its structurally related compounds (DDX) are difficult to degrade. Consequently, farmers are still frequently confronted with DDX contamination in their fields that was left over from the 1960s. This problem is particularly prevalent in contaminated soils that are intended to cultivate plants of the Cucurbitaceae family. These plants release so-called root exudates, which function as natural surfactants that mobilize the DDX present in the soils. Furthermore, surfactants are a common constituent of modern plant protection product (PPP) formulations, which can likewise cause DDX mobilization. The higher mobility of DDX caused by these surfactants can result in the absorption and accumulation of chlorinated pesticides in plants. The side effects of such surfactant-containing PPP formulations have historically been overlooked in the context of standard spraying protocols. The potential mobilization of DDX in soils and its accumulation in Cucurbita pepo due to the surfactants present in standard PPPs formulations was investigated using two field trials. One field was treated with a conventional PPP, while the other was treated with a biological PPP; a control field was left untreated, within which pumpkins were cultivated. Soil samples were taken before and after the application of PPP. The DDX content was subsequently determined in extracts from the soil phase samples and soil water fractions. The background DDX contamination of the soils was comparable in all three test fields. The comparative evaluation showed that the field treated with the biological PPP formulation exhibited a considerable increase in DDX mobility compared to the untreated and conventionally cultivated areas (Paper 1). An analysis of its respective water fraction revealed that it was more contaminated with DDX than the control treatments. This increase suggests a higher bioavailability that can be traced back to the presence of surfactants and oils in the PPP formulations (Paper 1). This higher bioavailability may have been accompanied by an increase in the DDX uptake of the cultivated plants. Furthermore, it was found that treatment with specific formulations of emulsifiable concentrates (EC) promoted DDX mobilization. This mobilizing effect was most likely due to the differing composition of the surfactant and proportions of oils in the PPPs. The second field test focused on differential DDX accumulation in Cucurbita pepo cv. Howden by different PPPs. Fields were treated with PPP in accordance with the official spraying plans and regulations set out by the Federal Ministry of Food and Agriculture (BMEL). Samples from the pumpkin plants roots, shoots, as well as the pumpkins themselves were taken during the cultivation period. The DDX content in the roots from the control fields and the fields with conventional PPP treatments remained virtually unchanged; however, the DDX content increased in the biologically treated area (Paper 2). The pumpkin shoots did not exhibit any increases in DDX concentration during the growing phase regardless of the field sampled. However, an increased DDX content was detected in the shoots of the plants in all test fields shortly before harvesting. At the end of the growing phase, fruits from the biologically treated area showed a higher DDX content than those from the control and conventionally treated areas. In addition, the most critical DDT metabolite, DDE, was found to have been transported to distant parts of the plant, while DDD was detectable in the roots and shoots but not in the fruits of the pumpkins (Paper 2). An assessment of the results of both experiments confirmed a direct correlation between DDX mobilization in the soil and plant uptake. In addition, the bioaccumulation factors of the biologically treated areas were markedly higher than those seen in the conventionally treated and control areas. The results of the field trials show that the mobilization of DDT, as well as the likely mobilization of other lipophilic contaminants, can become problematic for farmers using surfactant-containing EC formulations. However, this observation also provides opportunities for improved phytoremediation by applying EC formulations with high mobilization potentials. The field trials indicate that the mobilizing effects of DDT prompted by EC mixtures depend on the surfactant content in the PPP formulations as well as environmental conditions such as soil conditions, soil water content, and precipitation. Unravelling the optimal range of surfactant-rich formulations and environmental conditions could lead to a promising strategy for soil phytoremediation.
Identification of markers for dietary intake and health status by GC-MS based metabolite profiling approaches
(2020) Mack, Carina I.; Kulling, Sabine E.
Markers are compounds that can be used as indicators of an exposure, a metabolic state, or any other effect. Metabolomics and metabolite profiling approaches for marker discovery will increasingly gain significance. In the context of food, diet, and health, these approaches allow among others the identification of dietary intake markers, which can complement and verify traditional dietary assessment methods in epidemiologic studies. Consequently, the investigation of associations between diet and health status in general, and also in particular diet-related diseases will be improved. Compared to classical biomarker studies, metabolomics enables a more comprehensive investigation of clinical markers for diagnosis, prognosis and monitoring of diseases, such as type 2 diabetes mellitus. Especially, early diagnosis in pre-disease states, where symptoms are not yet evident, are of particular interest. The aim of this thesis was to evaluate the application of GC-MS based metabolite profiling approaches for the identification of markers for dietary intake and health status. In this respect, volatile organic compounds and sugar compounds were analyzed to discover marker candidates in urine and plasma samples from a cross-sectional study with 300 participants, as well as from a human intervention study with diabetic, prediabetic and healthy participants. In the past, the search for markers of dietary intake mostly focused on non-volatile metabolites. To explore the potential of the volatilome, urine samples of a cross-sectional study were analyzed aiming to exemplary identify markers of coffee consumption using an untargeted HS-SPME-GC×GC-MS method. Six marker candidates were identified from a profile of 138 volatile organic compounds with the most robust represented by 3,4-dimethyl-2,5-furandione. Moreover, the correlation with the general dietary intake data highlighted the volatilome as a particularly interesting source for the detection of new dietary markers. The chromatographic separation of sugar compounds is often insufficient due to the high structural similarities. Therefore, in most studies common and well-known sugar compounds are analyzed in human body fluids. Within the scope of this thesis, a semitargeted GC-MS sugar profiling method was developed, which revealed a more complex sugar profile, both in urine and plasma, than described so far or expected. Rare sugar compounds such as psicose and trehalose were detected. However, the knowledge about their origin and presence in urine or plasma is limited to date. Moreover, the maltose concentration in urine was shown to be dependent on sex and menopause status (pre- and post-menopausal) – a relationship with the vaginal microbiota is suggested here. In addition, the association of the urinary sugar profile with dietary intake data enabled the identification and confirmation of several new and also known marker candidates as for example, for consumption of avocado, dairy products and alcohol. The plasma sugar profiles of healthy, prediabetic and diabetic volunteers after an oral glucose tolerance test could be clearly distinguished, independent of glucose. Remarkably, a variety of sugar compounds showed marked postprandial differences dependent on health status. For example, trehalose showed a profile similar to the insulin-dependent profile of glucose. However, the origin and underlying biological mechanism for those sugar compounds remain to be elucidated. During the application of the one-dimensional GC-MS sugar profiling method to urine and plasma samples, it became evident that even more sugar compounds might be present, although in low concentrations, but were not detected due to limitations of the analytical method. Therefore, the one-dimensional method was transferred into a two-dimensional GC×GC-MS method. Improved sensitivity and separation finally enabled the detection of 84 instead of 55 sugar compounds in urine. The two-dimensional method was applied in an intervention study with apples, and revealed marker candidates for apple consumption for future validation. Overall, the results illustrate the benefit of a comprehensive analysis of sugar compounds in urine and plasma, including minor and rare sugar derivatives. The GC-MS based metabolite profiling approaches addressing the volatilome and the sugar profile, respectively, were demonstrated to be promising approaches for the identification of markers for dietary intake and health status. Future work should address the identification of unknown compounds, the adaptation of the GC×GC-MS sugar profiling method for quantitative purposes, and especially the validation of the identified marker candidates with respect to their suitability to more accurately assess dietary intake or diabetic state. High priority should also be given to the biochemical mechanisms and the origin of the compounds as well as their physiological or pathophysiological function in human metabolism.

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